1. Field of the Invention
The present invention relates to the field of engineered peptides, and to the field of peptides which bind to integrins, and particularly to integrin binding peptides that bind to platelet integrins and methods of using the peptides in anti-thrombotic therapies.
2. Related Art
Development of highly specific protein ligands that selectively target a single member in a family of closely related receptors has long been a significant molecular engineering problem. Integrin receptors present a particular challenge because recognition of many family members is mediated by an Arg-Gly-Asp (RGD) consensus sequence. Integrins are a class of diverse heterodimeric (α/β) receptors that are involved in cell adhesion to the extracellular matrix and mediate signaling pathways involved in cell cycle progression. As a result, several family members have generated much interest as potential therapeutic targets in the biomedical and pharmaceutical arenas. Integrins αIIbβ3 and αvβ3 are important clinical targets for prevention of platelet-mediated thrombosis and tumor angiogenesis, respectively; however, the high degree of similarity between these two receptors, as well as the αvβ5 integrin, has made it challenging to generate RGD-containing protein ligands that selectively target only a single integrin with high affinity.
An emerging approach for developing novel protein ligands is to use a naturally-occurring protein as a framework, or scaffold, and introducing amino acid mutations that confer recognition to a specific molecular target. Directed evolution is a powerful combinatorial technique for engineering new molecular recognition properties into protein scaffolds, often with remarkable affinities and specificities. By replacing loops or domains with random or rationally designed mutations that sample a large diversity of amino acid sequence space and isolating variants that possess desired molecular recognition properties, new proteins have been generated that bind diverse targets. Ideal protein scaffolds provide a stable, well-structured core and solvent-exposed loops or domains that are highly tolerant to substitution. Protein scaffolds that have been successfully evolved to bind new targets include fibronectin, A-domains, anticalins, ankyrin repeats, and cystine knots, among others.
Cystine knot (knottin) scaffolds have been used previously in other applications that require rapid biodistribution and short in vivo half lives, such as imaging in living subjects, and offer promise as oral peptide drugs. Knottins are a diverse class of small, highly structured polypeptides with up to 60 amino acids in length and possessing a core domain of three or more interwoven disulfide bonds. The structural rigidity conferred by the disulfide-bonded knottin framework leads to exceptionally high thermal and proteolytic stability, and the solvent-exposed loops spanned by these disulfide bonds are moderately to highly tolerant of mutations. Much of the development of knottin as protein-engineering scaffolds have focused on two family members: the Ecballium elaterium trypsin inhibitor II (EETI-II) and the melanocortin receptor binding domain of the human Agouti-related protein (AgRP). EETI-II contains three disulfide bonds, while AgRP contains five; therefore several truncated versions of AgRP have been developed to simplify the scaffold. In one study, EETI-II and AgRP have been used as scaffolds to introduce entire grafted loops derived from snake venom disintegrins that contain RGD or KGD integrin-recognition sequences resulting in knottin peptides that inhibit αIIbβ3, integrin-mediated platelet aggregation with half-maximal inhibitory concentration (IC50) values in the micromolar range. This study demonstrated that the structural confirmation of the scaffold and residues flanking the RGD sequence are critically important for the biological activity of the engineered peptides and also suggested that judicious selection of these neighboring residues might be an effective strategy by which to generate knottin peptides with enhanced potency.
Previously, EETI-II and AgRP knottins that bind to integrin αvβ3 (the vitronectin receptor) with low- to sub-nanomolar affinities were engineered. In both of these prior studies, peptide mutants that bound αvβ3 integrin were identified from yeast-displayed libraries where a single knottin loop was substituted with a loop containing an RGD motif, and randomized flanking residues. Surprisingly, although the library screens were performed only against αvβ3 integrin, the two engineered scaffolds showed very different integrin specificities. In addition to binding αvβ3 integrin with relative affinities of 10-30 nM, the engineered EETI-II peptides also bound with low nanomolar affinity to the related αvβ5 integrin. Moreover, one peptide was found to bind with, high affinity to α5β1 integrin (the fibronectin receptor) as well as to αvβ3 and αvβ5 integrins. None of these engineered EETI-II peptides bound αIIbβ3 integrin with affinities stronger than the micromolar range. In contrast, the engineered AgRP peptides, bound αvβ3 integrin with high affinity (KD˜1-10 nM) but did not appreciably bind to αvβ5 or α5β1 integrins. Additionally, the AgRP peptides weakly bound αIIbβ3 integrin (KD values could not be accurately determined, due to low affinity, but were estimated at greater than several hundred nM). The specificity of the engineered AgRP peptides for αvβ3 integrin was intriguing in light of previous challenges in developing protein scaffolds that could selectively target αvβ3 integrin with high affinity over αvβ5 and αIIbβ3 integrins. For example, when phage-displayed libraries of tendamistat analogs were screened for variants that bound to αvβ3, αvβ5, or αIIbβ3 integrins, most of the proteins selected bound at least two of these integrins. A common feature in these studies and others is that the conformation of the RGD motif is critical in determining both the affinity and specificity of the ligand-integrin interaction. Accordingly, improved integrin binding affinities and specificities have been achieved with cyclic and highly structured peptides relative to linear peptides. Similarly, the residues flanking the RGD motif have a significant role in determining how the recognition sequence is presented to integrin receptors; thus higher affinities and specificities also have often been achieved when the RGD flanking residues were engineered for optimal binding, using combinational methods, as opposed to simple loop grafting of a sequence from a natural RGD-containing ligand.
To further explore the integrin specificities that can be achieved with engineered cystine-knot peptides, and to expand the repertoire of available integrin-targeting molecules, we sought to determine whether a truncated form of AgRP and AgTx (Agatoxin) which have a C-terminal portion removed, could serve as a scaffold for selectively binding integrins other than αvβ3. Engineered AgRP peptides that selectively bind αIIbβ3 integrin with high affinity versus αIIbβ3 could also provide insights into the mode of binding between integrins and conformationally-restricted scaffold loops. Moreover, such peptides would have therapeutic potential as αIIbβ3 integrin plays a critical role in the aggregation of platelets and is a clinically validated target for thrombosis. Despite the successes of three FDA-approved anti-αIIbβ3 drugs for precautionary or responsive treatment to ischemic complications, there remains an interest in improved αIIbβ3 integrin inhibitors to prevent platelet-aggregation.
Presented below is background information on certain aspects of the present invention as they may relate to technical features referred to in the detailed description, but not necessarily described in detail. That is, individual parts or methods used in the present invention may be described in greater detail in the materials discussed below, which materials may provide further guidance to those skilled in the art for making or using certain aspects of the present invention as claimed. The discussion below should not be construed as an admission as to the relevance of the information to any claims herein or the prior art effect of the material described.